The mechanism of genetic repression by heterochromatin is a major unsolved problem in cellular and developmental gene regulation. Similar chromosomal regions with the features of heterochromatin are found at the telomeres in both complex and simple eukaryotes. Recently, an important clue to the understanding of this form of silencing has come from the discovery that certain histone N termini interact genetically and biochemically with specific Sir repressor proteins to repress and localize heterochromatin at the nuclear periphery in yeast. The goal of this proposal is to use genetic and biochemical means to determine how histones and non-histone proteins interact to allow silencing. Individual sites in histones H3 and H4 at both N terminal and non-N terminal regions involved in silencing will be mapped. These will be used genetically to identify non-histone factors which interact with thee sites to allow silencing. These sites will also be investigated as to their effect on the localization of telomeres to the nuclear periphery in order to determine whether the repression of heterochromatin is linked to its peripheral localization. It has been shown that histones H3 and H4 interact with the Sir3p and Sir4p repressors in vitro. In addition, Sir3p and Sir4p interact with each other. These interactions will be investigated to determine the sites of interaction and how they relate to the sites recognized by other silencing factors including Rap1, Sir3 and Sir4 proteins. This information will be used to correlate the interaction in vitro with defects in silencing in vivo. Another goal of this project is to determine biochemically, with crosslinking experiments, whether Sir3p and Sir4p interact directly with chromatin and specific histones in silenced regions. Factors responsible for these interactions will be identified in these experiments by using mutations in proteins known to be involved in silencing. Finally, histone acetyltransferases and deacetylases will be purified and their sequences will be used to clone the genes coding for these enzymes to determine genetically and biochemically whether acetylation of specific histone lysine residues is a means for regulating heterochromatin structure. These studies, which are designed to define the components which interact to form the heterochromatic complex in yeast, may lead to a novel understanding of the molecular nature of heterochromatin in eukaryotic organisms.